PROJECT SUMMARY Cas9 is an RNA-guided DNA endonuclease that is being used for sequence-specific DNA recognition, genome engineering, targeted gene activation/repression and genome imaging. Two commonly used variants of Cas9 are SpCas9 and SaCas9, which naturally occur in S pyogenes and S aureus, respectively. A pressing concern in the therapeutic genome editing using Cas9 is undesired off-target editing and chromosomal translocations that are associated with high activity levels of Cas9. Much interest also exists for rapid termination of Cas9 activity upon completion of on-target editing. Additionally, rapid, dosable, reversible, temporal, and orthogonal control of Cas9-based technologies (e.g., transcriptional activation and repression) will significantly expand the application of these technologies in biomedical research. We propose to develop small-molecule inhibitors of SpCas9 and SaCas9 that will allow rapid, dosable, temporal, and orthogonal control of Cas9 activities in HIV-associated immune cells as a model. To this end, we propose to deploy state-of-art technologies from organic chemistry and chemical informatics, biophysics, structural biology, and genome engineering.